pB-dCas9-PBE

LOCUS       pB-dCas9-PBE              17950 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expresses CRISPR base editor in maize.
ACCESSION   .
VERSION     .
KEYWORDS    pB-dCas9-PBE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17950)
  AUTHORS   Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C
  TITLE     Precise base editing in rice, wheat and maize with a Cas9-cytidine 
            deaminase fusion.
  JOURNAL   Nat Biotechnol. 2017 May;35(5):438-440. doi: 10.1038/nbt.3811. Epub 
            2017 Feb 27.
  PUBMED    28244994
REFERENCE   2  (bases 1 to 17950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17950
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    21..45
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     233..255
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     247..264
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     248..264
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        331..710
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     CDS             990..1781
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        1933..2008
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2312..4304
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             4315..4335
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4336..5019
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             5071..9171
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9172..9219
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9241..9489
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             9502..9522
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(10180..10196)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10180..10196)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10193..10215)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10204..10220
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(10228..10258)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10273..10294
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        10485..10830
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     10773..10793
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             10881..11432
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     polyA_signal    11439..11613
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11691..11715
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             12140..12934
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      13021..13609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13510..13529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13763..13780
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13795..13935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13861..13880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     14021..14043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      14279..14473
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(14539..15612)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(16041..16670)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta
       61 aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc
      121 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      181 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      241 cgacgttgta aaacgacggc cagtgccaag cttagtaatt catccaggtc tccaagttct
      301 aggattttca gaactgcaac ttattttatc aaggaatctt taaacatacg aacagatcac
      361 ttaaagttct tctgaagcaa cttaaagtta tcaggcatgc atggatcttg gaggaatcag
      421 atgtgcagtc agggaccata gcacaagaca ggcgtcttct actggtgcta ccagcaaatg
      481 ctggaagccg ggaacactgg gtacgttgga aaccacgtga tgtgaagaag taagataaac
      541 tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt attgcagtat
      601 gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc acctcggcta
      661 tccacataga tcaaagctga tttaaaagag ttgtgcagat gatccgtggc gtgagaccaa
      721 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
      781 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
      841 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
      901 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
      961 gcagtcgccc taaaacaaag ttaaacatca tgggggaagc ggtgatcgcc gaagtatcga
     1021 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     1081 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     1141 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     1201 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     1261 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     1321 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     1381 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     1441 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     1501 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     1561 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     1621 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     1681 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     1741 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctagct agaaattcgt
     1801 tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg ttagatgcac taagcacata
     1861 attgctcaca gccaaactat caggtcaagt ctgcttttat tatttttaag cgtgcataat
     1921 aagccggtct cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1981 acttgaaaaa gtggcaccga gtcggtgctt ttttttttcg ttttgcattg agttttctcc
     2041 gtcgcatgtt tgcagtttta ttttccgttt tgcattgaaa tttctccgtc tcatgtttgc
     2101 agcgtgttca aaaagtacgc agctgtattt cacttattta cggcgccaca ttttcatgcc
     2161 gtttgtgcca actatcccga gctagtgaat acagcttggc ttcacacaac actggtgacc
     2221 cgctgacctg ctcgtacctc gtaccgtcgt acggcacagc atttggaatt aaagggtgtg
     2281 atcgatactg cttgctgcta agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc
     2341 ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt
     2401 tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa actttactct
     2461 acgaataata taatctatag tactacaata atatcagtgt tttagagaat catataaatg
     2521 aacagttaga catggtctaa aggacaattg agtattttga caacaggact ctacagtttt
     2581 atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac
     2641 ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa
     2701 tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct
     2761 attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa
     2821 attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag
     2881 tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc
     2941 agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg
     3001 acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt
     3061 gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc
     3121 accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc
     3181 gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca
     3241 cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc
     3301 cgctcgtcct cccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt
     3361 tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc
     3421 cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa
     3481 cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat
     3541 cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt
     3601 caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt
     3661 gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact
     3721 acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg
     3781 aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt
     3841 tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg
     3901 ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt
     3961 tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg
     4021 atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac
     4081 atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat
     4141 aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc
     4201 agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt
     4261 tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcagccctag gatgccaaag
     4321 aagaagagga aggtttcatc ggagaccggc cctgttgctg ttgaccccac cctgcggcgg
     4381 agaatcgagc cacacgagtt cgaggtgttc ttcgacccaa gggagctccg caaggagacg
     4441 tgcctcctgt acgagatcaa ctggggcggc aggcactcca tctggaggca caccagccaa
     4501 aacaccaaca agcacgtgga ggtcaacttc atcgagaagt tcaccaccga gaggtacttc
     4561 tgcccaaaca cccgctgctc catcacctgg ttcctgtcct ggagcccatg cggcgagtgc
     4621 tccagggcca tcaccgagtt cctcagccgc tacccacacg tcaccctgtt catctacatc
     4681 gccaggctct accaccacgc cgacccaagg aacaggcagg gcctccgcga cctgatctcc
     4741 agcggcgtga ccatccaaat catgaccgag caggagtccg gctactgctg gaggaacttc
     4801 gtcaactact ccccaagcaa cgaggcccac tggccaaggt acccacacct ctgggtgcgc
     4861 ctctacgtgc tcgagctgta ctgcatcatc ctcggcctgc caccatgcct caacatcctg
     4921 aggcgcaagc aaccacagct gaccttcttc accatcgccc tccaaagctg ccactaccag
     4981 aggctcccac cacacatcct gtgggctacc ggcctcaagt ccggcagcga gacgccaggc
     5041 acctccgaga gcgctacgcc tgaacttaag gacaagaagt actcgatcgg cctcgccatc
     5101 gggacgaact cagttggctg ggccgtgatc accgacgagt acaaggtgcc ctctaagaag
     5161 ttcaaggtcc tggggaacac cgaccgccat tccatcaaga agaacctcat cggcgctctc
     5221 ctgttcgaca gcggggagac cgctgaggct acgaggctca agagaaccgc taggcgccgg
     5281 tacacgagaa ggaagaacag gatctgctac ctccaagaga ttttctccaa cgagatggcc
     5341 aaggttgacg attcattctt ccaccgcctg gaggagtctt tcctcgtgga ggaggataag
     5401 aagcacgagc ggcatcccat cttcggcaac atcgtggacg aggttgccta ccacgagaag
     5461 taccctacga tctaccatct gcggaagaag ctcgtggact ccaccgataa ggcggacctc
     5521 agactgatct acctcgctct ggcccacatg atcaagttcc gcggccattt cctgatcgag
     5581 ggggatctca acccagacaa cagcgatgtt gacaagctgt tcatccaact cgtgcagacc
     5641 tacaaccaac tcttcgagga gaacccgatc aacgcctctg gcgtggacgc gaaggctatc
     5701 ctgtccgcga ggctctcgaa gtccaggagg ctggagaacc tgatcgctca gctcccaggc
     5761 gagaagaaga acggcctgtt cgggaacctc atcgctctca gcctggggct caccccgaac
     5821 ttcaagtcga acttcgatct cgctgaggac gccaagctgc aactctccaa ggacacctac
     5881 gacgatgacc tcgataacct cctggcccag atcggcgatc aatacgcgga cctgttcctc
     5941 gctgccaaga acctgtcgga cgccatcctc ctgtcagata tcctccgcgt gaacaccgag
     6001 atcacgaagg ctccactctc tgcctccatg atcaagcgct acgacgagca ccatcaggat
     6061 ctgaccctcc tgaaggcgct ggtccgccaa cagctcccgg agaagtacaa ggagattttc
     6121 ttcgatcagt cgaagaacgg ctacgctggg tacatcgacg gcggggcctc acaagaggag
     6181 ttctacaagt tcatcaagcc aatcctggag aagatggacg gcacggagga gctcctggtg
     6241 aagctcaaca gggaggacct cctgcggaag cagagaacct tcgataacgg cagcatcccc
     6301 caccaaatcc atctcgggga gctgcacgcc atcctgagaa ggcaagagga cttctaccct
     6361 ttcctcaagg ataaccggga gaagatcgag aagatcctga ccttcagaat cccatactac
     6421 gtcggccctc tcgcgcgggg gaactcaaga ttcgcttgga tgacccgcaa gtctgaggag
     6481 accatcacgc cgtggaactt cgaggaggtg gtggacaagg gcgctagcgc tcagtcgttc
     6541 atcgagagga tgaccaactt cgacaagaac ctgcccaacg agaaggtgct ccctaagcac
     6601 tcgctcctgt acgagtactt caccgtctac aacgagctca cgaaggtgaa gtacgtcacc
     6661 gagggcatgc gcaagccagc gttcctgtcc ggggagcaga agaaggctat cgtggacctc
     6721 ctgttcaaga ccaaccggaa ggtcacggtt aagcaactca aggaggacta cttcaagaag
     6781 atcgagtgct tcgattcggt cgagatcagc ggcgttgagg accgcttcaa cgccagcctc
     6841 gggacctacc acgatctcct gaagatcatc aaggataagg acttcctgga caacgaggag
     6901 aacgaggata tcctggagga catcgtgctg accctcacgc tgttcgagga cagggagatg
     6961 atcgaggagc gcctgaagac gtacgcccat ctcttcgatg acaaggtcat gaagcaactc
     7021 aagcgccgga gatacaccgg ctgggggagg ctgtcccgca agctcatcaa cggcatccgg
     7081 gacaagcagt ccgggaagac catcctcgac ttcctgaaga gcgatggctt cgccaacagg
     7141 aacttcatgc aactgatcca cgatgacagc ctcaccttca aggaggatat ccaaaaggct
     7201 caagtgagcg gccaggggga ctcgctgcac gagcatatcg cgaacctcgc tggctccccc
     7261 gcgatcaaga agggcatcct ccagaccgtg aaggttgtgg acgagctcgt gaaggtcatg
     7321 ggccggcaca agcctgagaa catcgtcatc gagatggcca gagagaacca aaccacgcag
     7381 aaggggcaaa agaactctag ggagcgcatg aagcgcatcg aggagggcat caaggagctg
     7441 gggtcccaaa tcctcaagga gcacccagtg gagaacaccc aactgcagaa cgagaagctc
     7501 tacctgtact acctccagaa cggcagggat atgtacgtgg accaagagct ggatatcaac
     7561 cgcctcagcg attacgacgt cgatgctatc gttccccagt ctttcctgaa ggatgactcc
     7621 atcgacaaca aggtcctcac caggtcggac aagaaccgcg gcaagtcaga taacgttcca
     7681 tctgaggagg tcgttaagaa gatgaagaac tactggaggc agctcctgaa cgccaagctg
     7741 atcacgcaaa ggaagttcga caacctcacc aaggctgaga gaggcgggct ctcagagctg
     7801 gacaaggccg gcttcatcaa gcggcagctg gtcgagacca gacaaatcac gaagcacgtt
     7861 gcgcaaatcc tcgactctcg gatgaacacg aagtacgatg agaacgacaa gctgatcagg
     7921 gaggttaagg tgatcaccct gaagtctaag ctcgtctccg acttcaggaa ggatttccag
     7981 ttctacaagg ttcgcgagat caacaactac caccatgccc atgacgctta cctcaacgct
     8041 gtggtcggca ccgctctgat caagaagtac ccaaagctgg agtccgagtt cgtgtacggg
     8101 gactacaagg tttacgatgt gcgcaagatg atcgccaagt cggagcaaga gatcggcaag
     8161 gctaccgcca agtacttctt ctactcaaac atcatgaact tcttcaagac cgagatcacg
     8221 ctggccaacg gcgagatccg gaagagaccg ctcatcgaga ccaacggcga gacgggggag
     8281 atcgtgtggg acaagggcag ggatttcgcg accgtccgca aggttctctc catgccccag
     8341 gtgaacatcg tcaagaagac cgaggtccaa acgggcgggt tctcaaagga gtctatcctg
     8401 cctaagcgga acagcgacaa gctcatcgcc agaaagaagg actgggaccc aaagaagtac
     8461 ggcgggttcg acagccctac cgtggcctac tcggtcctgg ttgtggcgaa ggttgagaag
     8521 ggcaagtcca agaagctcaa gagcgtgaag gagctcctgg ggatcaccat catggagagg
     8581 tccagcttcg agaagaaccc aatcgacttc ctggaggcca agggctacaa ggaggtgaag
     8641 aaggacctga tcatcaagct cccgaagtac tctctcttcg agctggagaa cggcaggaag
     8701 agaatgctgg cttccgctgg cgagctccag aaggggaacg agctcgcgct gccaagcaag
     8761 tacgtgaact tcctctacct ggcttcccac tacgagaagc tcaagggcag cccggaggac
     8821 aacgagcaaa agcagctgtt cgtcgagcag cacaagcatt acctcgacga gatcatcgag
     8881 caaatctccg agttcagcaa gcgcgtgatc ctcgccgacg cgaacctgga taaggtcctc
     8941 tccgcctaca acaagcaccg ggacaagccc atcagagagc aagcggagaa catcatccat
     9001 ctcttcaccc tgacgaacct cggcgctcct gctgctttca agtacttcga caccacgatc
     9061 gatcggaaga gatacacctc cacgaaggag gtcctggacg cgaccctcat ccaccagtcg
     9121 atcaccggcc tgtacgagac gaggatcgac ctctcacaac tcggcgggga taagagaccc
     9181 gcagcaacca agaaggcagg gcaagcaaag aagaagaaga cgcgtgactc cggcggcagc
     9241 accaacctgt ccgacatcat cgagaaggag acgggcaagc aactcgtgat ccaggagagc
     9301 atcctcatgc tgccagagga ggtggaggag gtcatcggca acaagccaga gtccgacatc
     9361 ctggtgcaca ccgcctacga cgagtccacc gacgagaacg tcatgctcct gaccagcgac
     9421 gccccagagt acaagccatg ggccctcgtc atccaggaca gcaacgggga gaacaagatc
     9481 aagatgctgt cgggggggag cccaaagaag aagcggaagg tgtagagctc agagctttcg
     9541 ttcgtatcat cggtttcgac aacgttcgtc aagttcaatg catcagtttc attgcgcaca
     9601 caccagaatc ctactgagtt tgagtattat ggcattggga aaactgtttt tcttgtacca
     9661 tttgttgtgc ttgtaattta ctgtgttttt tattcggttt tcgctatcga actgtgaaat
     9721 ggaaatggat ggagaagagt taatgaatga tatggtcctt ttgttcattc tcaaattaat
     9781 attatttgtt ttttctctta tttgttgtgt gttgaatttg aaattataag agatatgcaa
     9841 acattttgtt ttgagtaaaa atgtgtcaaa tcgtggcctc taatgaccga agttaatatg
     9901 aggagtaaaa cacttgtagt tgtaccatta tgcttattca ctaggcaaca aatatatttt
     9961 cagacctaga aaagctgcaa atgttactga atacaagtat gtcctcttgt gttttagaca
    10021 tttatgaact ttcctttatg taattttcca gaatccttgt cagattctaa tcattgcttt
    10081 ataattatag ttatactcat ggatttgtag ttgagtatga aaatattttt taatgcattt
    10141 tatgacttgc caattgattg acaacgaatt cgtaatcatg tcatagctgt ttcctgtgtg
    10201 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    10261 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    10321 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
    10381 cggtttgcgt attggctaga gcagcttgcc aacatggtgg agcacgacac tctcgtctac
    10441 tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa
    10501 agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa
    10561 aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa aggaaaggct
    10621 atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc
    10681 atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc
    10741 tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata
    10801 taaggaagtt catttcattt ggagaggaca cgctgaaatc accagtctct ctctacaaat
    10861 ctatctctct cgagtctacc atgagcccag aacgacgccc ggccgacatc cgccgtgcca
    10921 ccgaggcgga catgccggcg gtctgcacca tcgtcaacca ctacatcgag acaagcacgg
    10981 tcaacttccg taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc
    11041 gggagcgcta tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg
    11101 cgggcccctg gaaggcacgc aacgcctacg actggacggc cgagtcgacc gtgtacgtct
    11161 ccccccgcca ccagcggacg ggactgggct ccacgctcta cacccacctg ctgaagtccc
    11221 tggaggcaca gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg
    11281 tgcgcatgca cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg gccggcttca
    11341 agcacgggaa ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc
    11401 cccgtccggt cctgcccgtc accgagattt gactcgagtt tctccataat aatgtgtgag
    11461 tagttcccag ataagggaat tagggttcct atagggtttc gctcatgtgt tgagcatata
    11521 agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc
    11581 ctaaaaccaa aatccagtac taaaatccag atcccccgaa ttaattcggc gttaattcag
    11641 tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca
    11701 caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca
    11761 ccactcgata caggcagccc atcagtccgg gacggcgtca gcgggagagc cgttgtaagg
    11821 cggcagactt tgctcatgtt accgatgcta ttcggaagaa cggcaactaa gctgccgggt
    11881 ttgaaacacg gatgatctcg cggagggtag catgttgatt gtaacgatga cagagcgttg
    11941 ctgcctgtga tcaccgcggt ttcaaaatcg gctccgtcga tactatgtta tacgccaact
    12001 ttgaaaacaa ctttgaaaaa gctgttttct ggtatttaag gttttagaat gcaaggaaca
    12061 gtgaattgga gttcgtcttg ttataattag cttcttgggg tatctttaaa tactgtagaa
    12121 aagaggaagg aaataataaa tggctaaaat gagaatatca ccggaattga aaaaactgat
    12181 cgaaaaatac cgctgcgtaa aagatacgga aggaatgtct cctgctaagg tatataagct
    12241 ggtgggagaa aatgaaaacc tatatttaaa aatgacggac agccggtata aagggaccac
    12301 ctatgatgtg gaacgggaaa aggacatgat gctatggctg gaaggaaagc tgcctgttcc
    12361 aaaggtcctg cactttgaac ggcatgatgg ctggagcaat ctgctcatga gtgaggccga
    12421 tggcgtcctt tgctcggaag agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct
    12481 gtatgcggag tgcatcaggc tctttcactc catcgacata tcggattgtc cctatacgaa
    12541 tagcttagac agccgcttag ccgaattgga ttacttactg aataacgatc tggccgatgt
    12601 ggattgcgaa aactgggaag aagacactcc atttaaagat ccgcgcgagc tgtatgattt
    12661 tttaaagacg gaaaagcccg aagaggaact tgtcttttcc cacggcgacc tgggagacag
    12721 caacatcttt gtgaaagatg gcaaagtaag tggctttatt gatcttggga gaagcggcag
    12781 ggcggacaag tggtatgaca ttgccttctg cgtccggtcg atcagggagg atatcgggga
    12841 agaacagtat gtcgagctat tttttgactt actggggatc aagcctgatt gggagaaaat
    12901 aaaatattat attttactgg atgaattgtt ttagtaccta gaatgcatga ccaaaatccc
    12961 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
    13021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
    13081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
    13141 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
    13201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
    13261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
    13321 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
    13381 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
    13441 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
    13501 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
    13561 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
    13621 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
    13681 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
    13741 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
    13801 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
    13861 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
    13921 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
    13981 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
    14041 aggttttcac cgtcatcacc gaaacgcgcg aggcagggtg ccttgatgtg ggcgccggcg
    14101 gtcgagtggc gacggcgcgg cttgtccgcg ccctggtaga ttgcctggcc gtaggccagc
    14161 catttttgag cggccagcgg ccgcgatagg ccgacgcgaa gcggcggggc gtagggagcg
    14221 cagcgaccga agggtaggcg ctttttgcag ctcttcggct gtgcgctggc cagacagtta
    14281 tgcacaggcc aggcgggttt taagagtttt aataagtttt aaagagtttt aggcggaaaa
    14341 atcgcctttt ttctctttta tatcagtcac ttacatgtgt gaccggttcc caatgtacgg
    14401 ctttgggttc ccaatgtacg ggttccggtt cccaatgtac ggctttgggt tcccaatgta
    14461 cgtgctatcc acaggaaaca gaccttttcg acctttttcc cctgctaggg caatttgccc
    14521 tagcatctgc tccgtacatt aggaaccggc ggatgcttcg ccctcgatca ggttgcggta
    14581 gcgcatgact aggatcgggc cagcctgccc cgcctcctcc ttcaaatcgt actccggcag
    14641 gtcatttgac ccgatcagct tgcgcacggt gaaacagaac ttcttgaact ctccggcgct
    14701 gccactgcgt tcgtagatcg tcttgaacaa ccatctggct tctgccttgc ctgcggcgcg
    14761 gcgtgccagg cggtagagaa aacggccgat gccgggatcg atcaaaaagt aatcggggtg
    14821 aaccgtcagc acgtccgggt tcttgccttc tgtgatctcg cggtacatcc aatcagctag
    14881 ctcgatctcg atgtactccg gccgcccggt ttcgctcttt acgatcttgt agcggctaat
    14941 caaggcttca ccctcggata ccgtcaccag gcggccgttc ttggccttct tcgtacgctg
    15001 catggcaacg tgcgtggtgt ttaaccgaat gcaggtttct accaggtcgt ctttctgctt
    15061 tccgccatcg gctcgccggc agaacttgag tacgtccgca acgtgtggac ggaacacgcg
    15121 gccgggcttg tctcccttcc cttcccggta tcggttcatg gattcggtta gatgggaaac
    15181 cgccatcagt accaggtcgt aatcccacac actggccatg ccggccggcc ctgcggaaac
    15241 ctctacgtgc ccgtctggaa gctcgtagcg gatcacctcg ccagctcgtc ggtcacgctt
    15301 cgacagacgg aaaacggcca cgtccatgat gctgcgacta tcgcgggtgc ccacgtcata
    15361 gagcatcgga acgaaaaaat ctggttgctc gtcgcccttg ggcggcttcc taatcgacgg
    15421 cgcaccggct gccggcggtt gccgggattc tttgcggatt cgatcagcgg ccgcttgcca
    15481 cgattcaccg gggcgtgctt ctgcctcgat gcgttgccgc tgggcggcct gcgcggcctt
    15541 caacttctcc accaggtcat cacccagcgc cgcgccgatt tgtaccgggc cggatggttt
    15601 gcgaccgctc acgccgattc ctcgggcttg ggggttccag tgccattgca gggccggcag
    15661 gcaacccagc cgcttacgcc tggccaaccg cccgttcctc cacacatggg gcattccacg
    15721 gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc ctttagccgc taaaattcat
    15781 ctactcattt attcatttgc tcatttactc tggtagctgc gcgatgtatt cagatagcag
    15841 ctcggtaatg gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt gatcctccgc
    15901 cggcaactga aagttgaccc gcttcatggc tggcgtgtct gccaggctgg ccaacgttgc
    15961 agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc ttttgctcat
    16021 tttctcttta cctcattaac tcaaatgagt tttgatttaa tttcagcggc cagcgcctgg
    16081 acctcgcggg cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc ggcggcggca
    16141 gtgcctgggt agctcacgcg ctgcgtgata cgggactcaa gaatgggcag ctcgtacccg
    16201 gccagcgcct cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg cgacacgaca
    16261 aaggccgctt gtagccttcc atccgtgacc tcaatgcgct gcttaaccag ctccaccagg
    16321 tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg gaatcagcac gaagtcggct
    16381 gccttgatcg cggacacagc caagtccgcc gcctggggcg ctccgtcgat cactacgaag
    16441 tcgcgccggc cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat gccgacaacg
    16501 gttagcggtt gatcttcccg cacggccgcc caatcgcggg cactgccctg gggatcggaa
    16561 tcgactaaca gaacatcggc cccggcgagt tgcagggcgc gggctagatg ggttgcgatg
    16621 gtcgtcttgc ctgacccgcc tttctggtta agtacagcga taaccttcat gcgttcccct
    16681 tgcgtatttg tttatttact catcgcatca tatacgcagc gaccgcatga cgcaagctgt
    16741 tttactcaaa tacacatcac ctttttagac ggcggcgctc ggtttcttca gcggccaagc
    16801 tggccggcca ggccgccagc ttggcatcag acaaaccggc caggatttca tgcagccgca
    16861 cggttgagac gtgcgcgggc ggctcgaaca cgtacccggc cgcgatcatc tccgcctcga
    16921 tctcttcggt aatgaaaaac ggttcgtcct ggccgtcctg gtgcggtttc atgcttgttc
    16981 ctcttggcgt tcattctcgg cggccgccag ggcgtcggcc tcggtcaatg cgtcctcacg
    17041 gaaggcaccg cgccgcctgg cctcggtggg cgtcacttcc tcgctgcgct caagtgcgcg
    17101 gtacagggtc gagcgatgca cgccaagcag tgcagccgcc tctttcacgg tgcggccttc
    17161 ctggtcgatc agctcgcggg cgtgcgcgat ctgtgccggg gtgagggtag ggcgggggcc
    17221 aaacttcacg cctcgggcct tggcggcctc gcgcccgctc cgggtgcggt cgatgattag
    17281 ggaacgctcg aactcggcaa tgccggcgaa cacggtcaac accatgcggc cggccggcgt
    17341 ggtggtgtcg gcccacggct ctgccaggct acgcaggccc gcgccggcct cctggatgcg
    17401 ctcggcaatg tccagtaggt cgcgggtgct gcgggccagg cggtctagcc tggtcactgt
    17461 cacaacgtcg ccagggcgta ggtggtcaag catcctggcc agctccgggc ggtcgcgcct
    17521 ggtgccggtg atcttctcgg aaaacagctt ggtgcagccg gccgcgtgca gttcggcccg
    17581 ttggttggtc aagtcctggt cgtcggtgct gacgcgggca tagcccagca ggccagcggc
    17641 ggcgctcttg ttcatggcgt aatgtctccg gttctagtcg caagtattct actttatgcg
    17701 actaaaacac gcgacaagaa aacgccagga aaagggcagg gcggcagcct gtcgcgtaac
    17761 ttaggacttg tgcgacatgt cgttttcaga agacggctgc actgaacgtc agaagccgac
    17821 tgcactatag cagcggaggg gttggatcaa agtactttga tcccgagggg aaccctgtgg
    17881 ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt taaatatccg
    17941 attattctaa
//